H4k20me3

Immunostaining of heterochromatic and euchromatic regions was performed according to a standard protocol on separate cell samples. The cells were permeabilized with 0,2% Triton X-100 (Merck KGaA, Darmstadt, Germany) diluted in 1 x PBS (Life technologies GmbH, Darmstadt, Germany). After blocking with 2% bovine serum albumin (BSA) (Carl Roth GmbH, Karlsruhe, Germany) in 1x PBS the antibodies were diluted in 2% BSA blocking solution. The used primary antibodies were either directed against H3K4me3 (= mainly euchromatin regions) or H4K20me3 (= mainly constitutive heterochromatin regions) (Abcam, Cambridge, Great Britain). Secondary antibodies (Life technologies GmbH, Darmstadt, Germany) labelled with Alexa568 (λ_ex/em = 560 nm / 610 nm) were used for the fluorescence imaging. All samples were counterstained using DAPI (λ_ex/em = 364 nm / 454nm) and embedded in Prolong Gold (Life technologies GmbH, Darmstadt, Germany) before SPDM imaging.

The purified histones from serum and tissue that were resolved on 18% sodium duodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was either stained by silver staining method [19 (link)] or transferred to PVDF membrane, probed with site-specific modified histone antibodies, against H4K16Ac (Millipore#07-329), H4K20Me3 (Abcam#9053), γH2AX (Millipore#05-636), H3S10P (Millipore#06-570), H3K27Me3 (Millipore#07-449) and H3K9Me3 (Abcam#8898), and signals were detected by ECL plus detection kit (Millipore #WBKLS0500). Gel loading equivalence was done by silver staining method.

TCL and NCF, and histones were resolved on 10 and 18 % polyacrylamide SDS-PAGE, respectively, and transferred to PVDF membrane. Proteins on PVDF membrane were hybridized with anti-H3 (Upstate-06-755; 1:2000 dilution), H4 (Millipore-07-108; 1:4000 dilution), H3S10ph (Millipore-06-570; 1:7000 dilution), H4K16ac (Millipore-07-329; 1:8000 dilution), H4K20me3 (Abcam-9053; 1:4000 dilution), β-actin (Sigma-A5316; 1:10,000 dilution), MSK1 (Santacruz-9392; 1:2000 dilution), ph-MSK1 (Abcam-31190; 1:3000 dilution), ERK1/2 (Santacruz-292838; 1:2000 dilution), ph-ERK (Cell signaling-9910; 1:2000 dilution), p38 (Santacruz-728; 1:2000 dilution), ph-p38 (Cell signaling-9910; 1:2000 dilution), and anti-flag (Sigma-F3165; 1:5000 dilution). Signal was visualized using horseradish peroxidase-conjugated anti-rabbit/mouse secondary antibody and ECL plus chemiluminescence kit (Amersham). Wherever required, the densitometry analysis was done on immunoblot and membrane to determine their mean intensities using ImageJ software. For native proteins, mean intensity of immunoblot was normalized with the stained PVDF membrane; for phosphorylated forms, mean intensity of immunoblot was normalized with immunoblot of native proteins. The resulted value was used to express their mean relative levels in resection margin and tumor.

Western blotting was performed by standard procedures after tissue lysis in Laemmli buffer. Nitrocellulose blotted membranes were probed with the following antibodies: H3K27me (Abcam, ab6002), H3K9me3 (Abcam, ab8898), H4K20me3 (Abcam, ab9053), H3K4me3 (Abcam, ab8580), H3 (Cell Signalling), PCNA (GeneTex, GTX124496), γH2AX (Genetex, GTX127343), and α-Tubulin (Cell Signalling, 3873S). Development was performed by Odyssey CLX reader (LI-COR). Densitometry analysis was performed using LI-COR software. Signal intensity from three independent Western blots loaded with lysates derived from different individuals was used for densitometry calculations normalized to young samples.

Chromatin was isolated and immunoprecipitated using the Shearing ChIP Kit (Diagenode, C01020012, Denville, NJ, USA) and the OneDay ChIP kit (Diagenode, C01010080, Denville, NJ, USA), respectively, according to the manufacturer’s protocol. To immunoprecipitate H3K4me3 (Abcam, ab8580, Waltham, MA, USA), H3K9me3 (Abcam, ab8898, Waltham, MA, USA), H3K27me3 (Millipore, 07449, Darmstadt, Germany), H4K20me3 (Abcam, ab9053, Waltham, MA, USA), CTCF (Millipore, 07729, Darmstadt, Germany), and RNA Pol-II (Abcam, ab5408), we used 4 µg of each antibody and incubated them overnight. Immunoprecipitated DNA was analyzed by qPCR with the StepOne Real-time PCR system (Thermo Fisher Scientific, 4376600, Waltham, MA, USA) using specific primers for each locus. Total chromatin input from every sample was used as a reference for the comparative curves of the analyzed marks. Data were normalized against IgG.